The present invention relates to a novel approach to vary the production of gene products, particularly protein antigens, particularly in species of Bordetella, by changing the promoters of the genes coding for the respective proteins and altering their level of expression.
The bacterial species Bordetella comprises B. pertussis, B. parapertussis, B. bronchiseptica, and B. avium. The first two microorganisms are human pathogens, while the latter two are generally restricted to non-human hosts. B. pertussis and B. parapertussis cause the disease whooping cough with the former generating more severe symptoms. The disease, or vaccination against the disease (using an inactivated whole-cell vaccine), elicits antibodies against several antigens, typically pertussis toxin (PT), filamentous haemagglutinin (FHA), agglutinogens or fimbriae and the 69 kDa outer membrane protein or pertactin. These proteins represent the major immunogens that may be included, individually or in combination, in any vaccine used to protect against the disease, whether it be the inactivated whole-cell vaccine or a defined component vaccine. Therefore, the efficient expression of these antigens from the vaccine strain is crucial.
During the production of vaccine antigens by fermentation, it has been observed that FHA is secreted at approximately 7 and 10 times the molar level of pertactin and PT, respectively. While protein structural complexity and secretion efficiencies may be important factors influencing antigen yields, the level of expression of B. pertussis antigen genes also may be influenced by the relative strength and the regulation of their respective promoters. Thus, it may be possible to optimize antigen production by substituting autologous promoters, which may either increase or decrease the yield of selected antigens. The resulting B. pertussis strains would be more economical and better immunogens to use directly in whole-cell vaccines. In addition, promoter interchange also may represent a means to enhance fermentation and downstream processing efficiencies for component vaccines by altering the kinetics of production and yields of specific antigens.
Along with other genes, the pertussis toxin operon (TOX), the FHA operon, and the pertactin gene (PRN) are all positively regulated by the Bordetella virulence regulating gene (Bvg), formerly known as VIR. The nucleotide sequences of the TOX, FHA, and PRN structural genes and their promoters have been established and the corresponding protein sequences derived (see below). For the TOX operon, the Bvg responsive region of the promoter has been mapped to a position xe2x88x92170 bp from the start of transcription. The corresponding regulatory regions of the other genes have not yet been determined.
The use of killed whole-cell pertussis vaccines has resulted in a massive reduction in the incidence of whooping cough since their introduction in the 1950s. These vaccine preparations are efficacious but have been known for many years to be reactogenic and to be associated with local and systemic responses in vaccinees. There has thus been a great deal of effort to develop defined a cellular pertussis vaccines containing highly purified, well-characterized and non-reactogenic antigens. Such a cellular vaccines are used for immunization in Japan and are at various stages of clinical assessment in other countries. Defined pertussis vaccines consist of several combinations of the B. pertussis-specific antigens pertussis toxin (PT), filamentous hemagglutinin (FHA), the 69 kDa outer membrane protein (pertactin) and fimbrial agglutinogens. Replacing whole-cell whooping cough vaccines with the defined a cellular preparations has resulted in a substantial increase in the complexity and cost of vaccine manufacture. A major portion of these increased costs is due to the relatively low levels of PT and pertactin produced by B. pertussis strains, even when grown under optimized fermentation conditions. Increasing the level of antigen production by B. pertussis may be achieved by replacing the natural promoter for a gene encoding an antigen by another promoter as described in the present invention.
In accordance with the present invention, there is provided a novel method to alter protein expression in Bordetella species by substituting the promoter of one gene of a Bordetella species by that of another gene of a Bordetella species. It is possible to increase or decrease protein expression using this strategy as well as achieving sychronicity or asynchronicity of antigen production.
Accordingly, in one aspect, the present invention provides a hybrid Bordetella gene, comprising a Bordetella gene fused at an ATG start codon to an autologous Bordetella promoter. The Bordetella gene usually encodes an antigen.
Preferably, specific combinations of Bordetella genes and promoters are provided, including the TOX promoter in combination with the FHA or PRN genes; the FHA promoter in combination with the TOX or PRN genes; and the PRN promoter in combination with the TOX or FHA genes. In a particularly preferred embodiment, the Bordetella genes and promoters are those from Bordetella pertussis. 
The present invention further provides strains of Bordetella, particularly Bordetella pertussis, which contain the hybrid genes or multiples of such hybrid genes and which are capable of expression of a gene product of the Bordetella gene or genes. Such strains produce, upon culture, the gene product at a yield of production which is altered from the yield of production achieved by the same Bordetella strain containing a homologous gene comprising a Bordetella gene fused at an ATG start codon to its own native Bordetella promoter.
Specific new B. pertussis strains are described in the disclosure which follows, in particular those identified as B. pertussis strains Nos. 1290-4, 390-59, 590-473, 192-35, 192-10, 390-59, 590-11 and 890-49. In addition, B. pertussis strains from which at least one of the FHA gene and the PRN gene have been removed, particularly strain Nos. 390-101 and 1090-108-3, form other aspects of the invention. The invention further includes the plasmids useful in effecting transformation of the bordetella strain, particularly B. pertussis strains.
The gene products, usually the antigenic proteins, produced by culturing the Bordetella strain containing the hybrid gene generally are useful in vaccines against the disease of whooping cough (pertussis).
An aspect of the invention allows for the easier downstream separation of the resulting proteins, which makes the production of a component vaccine more economical. A further aspect of the invention allows for the preparation of a whole-cell pertussis vaccine in which, because of improved gene expression, antigenic proteins are distributed differently.
The technique described herein for expression of proteins from transformed strains of B. pertussis may be employed with other strains of Bordetella, such as B. parapertussis, B. bronchiseptica and B. avium. The technique also broadly is applicable to hybrid genes formed from any Bordetella gene and any native but autologous Bordetella promoter to effect expression of the desired gene product from a Bordetella strain containing the hybrid gene.
Accordingly, in yet another aspect of the present invention, there is provided a method of expression of a gene product from a Bordetella strain, which comprises forming a hybrid gene comprising a Bordetella gene fused at an ATG start codon to an autologous Bordetella promoter, introducing the hybrid gene into a Bordetella strain to form a transformed Bordetella strain, and culturing the transformed Bordetella strain to effect expression of a gene product encoded by the hybrid gene.
The disclsoure which follows specifically describes the invention with respect to hybrid genes containing promoters and genes of Bordetella pertussis, to Bordetella pertussis strains containing such hybrid genes, and to gene products of such strains. However, as will be apparent from the above discussion, the invention is broadly applicable to hybrid genes, strains and gene products of other Bordetella species, such as B. parapertussis, B. brochiseptica and B. Avium.